Tips & Tricks GPC/SEC: Branching Analysis

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1 Tips & Tricks GPC/SEC: Branching Analysis Daniela Held, Peter Montag, and Wolfgang Radke, PSS Polymer Standards Service GmbH, Mainz, Germany. Branching is one of the parameters chemists can adjust to produce polymer materials with optimized physical properties. Chromatography and advanced detection can help to characterize branched molecules. This instalment of Tips & Tricks explains more. Photo Credit: Justin Dominic Alessandro Guisseppe Abella / EyeEm/Getty Images An advantage of polymer materials is that their physical properties can be tailored for a specific application by adjusting many parameters. Besides composition, average molar mass, and the width of the molar mass distribution, another key parameter to control application properties is branching. Branching requires at least a single branch point where three or more chains are connected. Branching can occur as an undesired side reaction during synthesis or it can be introduced deliberately to optimize the physical properties of the material. Different routes exist to synthesize defined structures, such as star-shaped or comb-shaped molecules. The properties of branched polymers differ significantly from linear ones with respect to (melt) viscosity, glass transition temperature, the coefficient of bulk thermal expansion, solubility, and others. The property change depends on the parameters such as type of branching, length of the branches, and branching density. The characterization of complex mixtures comprising not only a molar mass distribution but branching distribution as well, represents a real challenge. Depending on the type of branching there are various detection and separation options that provide deeper insight. Gel permeation chromatography/ size-exclusion chromatography (GPC/SEC) hyphenated with on-line viscometry 1 (or less accurate multi-angle light scattering) can be used to characterize defined structures, such as star or comb-shaped polymers, or to investigate long chain branching. High temperature GPC (HT-GPC) with infrared (IR) detection can be used to investigate short-chain branching in polyolefins. For samples exhibiting broad molar mass distributions for branches and backbone

2 Figure 1: Mark-Houwink plot overlay of three different polyethylene samples. NBS-1475 (pink) is linear, linear low density polyethylene (LLDPE, black) has short-chain branching that does not influence the viscosity much, and low density polyethylene (LDPE, blue) exhibits long-chain branching, which leads to a reduction of viscosity. Viscosity Mark-Houwink-Plot and variations in branching density, the resolution of the size-based separation in GPC/SEC might not be sufficient to fully resolve the structures. Therefore alternative separation methods such as interaction chromatography (for example gradient polymer high performance liquid chromatography [HPLC] or temperature gradient interaction chromatography NBS-1475: lin. PE, pink, α = 0,68 LLDPE-C6: black, α = 0,70 LDPE: blue, α = 0, Universal calibration (Da) [TGIC]) or two-dimensional (D) chromatography should be applied. 3 GPC/SEC-Viscometry An on-line viscometer is classified as a molar mass sensitive detector, however, the signal intensity is dependent on the viscosity rather than on molar mass. Viscometers provide direct access to the density of the PSSWnGPC Unity, Buld 608, LAB_All 4, Instnz#1 molecules in solution. While the setup of such instrumentation, in most cases in combination with a light scattering detector, is very common, understanding the results and limitations requires more experience. The Mark Houwink plot is important for branching analysis; the logarithm of the intrinsic viscosity (obtained using on-line viscometry) is plotted versus the logarithm of the molar mass (obtained using universal calibration or light scattering detection). The slope of the Mark-Houwink plot (Mark-Houwink coefficient, α) is dependent on the shape of the molecule in solution. If the intrinsic viscosity (solid sphere) has no molar mass dependence a slope of 0 is expected, however, the Mark Houwink exponent of rigid rods is. Typical random coil polymers exhibit Mark-Houwink exponents in the range of 0.5 to 0.8, depending on solvent quality. Branching analysis can be straightforward, assuming that the data can be compared to a linear chain of identical chemical structure and molar mass. Figure 1 shows Mark Houwink plots for different polyethylene samples. A HT-GPC equipped with an on-line viscometer has been used to generate this plot. While the Mark Houwink plot of the linear low-density polyethylene (LLDPE), which has only short chain branching, nearly superimposes with the linear sample, High-Resolution Quantitative Characterization of Intact Biopharmaceuticals and Their Proteoforms ON-DEMAND WEBCAST Aired May 5, 016 Register for free at Proteins of biopharmaceutical interest are generally heterogeneous mixtures of proteoforms comprised of modifications. Accurate knowledge of the proteoform profile is critical for assessing the safety and stability of the drug. Current methods of analyzing post-translational modifications (PTMs) and glycan structures require proteolysis or glycan profiling that can result in lost information on correlated PTMs. Sample handling can also introduce artifacts and therefore, minimal sample preparation is desirable. Top-down mass spectrometry (MS) analysis of highly heterogeneous biopharmaceuticals is challenging due to the limited ability of current separation techniques in resolving proteoforms with small structural changes. In this work, we describe the top-down analysis of interferon-β1 (Avonex) in detail and preliminary data on middle-down (reduced) and intact mabs. Key Learning Objectives: Find out how CESI-MS enables high resolution intact separation and on-line top-down MS identification of PTMs with minimal sample preparation Learn how glycan isomers (such as the two isomers GF and 1 NANA) and deamidations can be resolved by intact analysis Learn how a similar approach was applied for intact and reduced analysis of mabs Who Should Attend Principal Investigators, department chairs, senior scientists, R&D directors, post-doctoral fellows, post-graduate researchers, and medical researchers. Presenter: David R. Bush, Ph.D. Scientific Manager Genedata, Inc. Moderator: Laura Bush Editorial Director LCGC 30 Minute Format Sponsored by Presented by For questions, contact Kristen Moore at kmoore@advanstar.com

3 Figure : Mark-Houwink for a star polymer using the arm-first approach. Arms with a narrow molar mass distribution are coupled to a core; the molar mass increase is a result of the addition of more arms to the core. The structure change (linear coil to a more and more dense sphere) is reflected by a maximum in intrinsic viscosity. Specific viscosity Intrinisc Viscosity Number of Arms Degree of Branching 1.1% Residual Arm the low-density polyethylene (LDPE), which comprises long-chain branching, deviates significantly. At the same molecular weight the LDPE chains reveal a significantly lower intrinsic viscosity compared to the linear sample. This is a consequence of branches being present. The deviation increases with increasing branching density. By extrapolating the Mark Houwink plots Star Polymer with average arm number 5 4.5% Dimer (still linear) 5 * * *10 5 1*10 6 Molar mass from universal calibration curve (D) Intr. Visc. Linear Counterpart 4.0% Coupled Star Polymer (H-shaped) of the branched and linear polymer to a common intercept, it is possible to detect the molar mass at which branching first occurs. By taking the ratio of the intrinsic viscosity of the branched and linear polymer at the same molar mass, it is possible to determine the contraction factor, g, from which conclusions on the number of branches can be deduced. PSS WinGPC Unity Figure shows the results of GPC/ SEC-viscometry of a poly(tert-butyl acrylate), PtBuA, star polymer. Star polymers are relatively simple branched polymers because they consist of several arms (linear chains) connected to a central core. The star polymer was synthesized using the arm-first approach. PtBuA arms of narrow molar mass distribution have been coupled using a small amount of a bifunctional cross-linker to form the core. This means that the molar mass increase of the star was obtained by coupling an increasing number of arms of approximately the same length to the core. The Mark Houwink plot reveals a maximum for the intrinsic viscosity, which has also been observed for dendrimers. Starting with a linear precursor, the coupling of two linear chains forms a still linear molecule, the dimer. Further reaction of precursor molecules with the core leads to three-arm and higher arm star polymers. Here the increase of the intrinsic viscosity with molar mass is counterbalanced by the decreasing intrinsic viscosity resulting from an increasing segment density with increasing number of arms for the branched structures. This variation in molecular structure can be nicely monitored from the viscosity measured using an on-line viscometer. LIVE WEBCAST: Wednesday, June 15, am PDT 10 am CDT 11 am EDT 4 pm BST 5 pm CEST Register free at: EVENT OVERVIEW The screening and routine quantitation of pesticide residues in food products is one of the most important and demanding applications in food safety. Despite the recent technological advancements in LC-MS, it is still challenging to quantify hundreds of LC-amenable pesticides with a robust, sensitive workflow solution. This presentation describes the development and implementation of complete workflow solutions based on LC-MS/MS and LC-HRAM-MS/MS. These ready-to-go solutions have been validated in three matrices across four different laboratories. In addition, customized software used for data acquisition and processing allows the users to rapidly implement these methods and enhance productivity. Key Learning Objectives Address critical challenges in targeted or untargeted quantitation of pesticides in food laboratories using either triple quadrupole MS or high-resolution accurate mass (HRAM) MS instrumentation Learn about robust, routine workflows that can increase laboratory and organizational productivity Who Should Attend Researchers and analysts in need of fast and cost-effective solutions for the analysis of pesticides in food Presenters Pesticide Residues Analysis Webinar Comprehensive Pesticide Quantitation Workflow with LC-MS Ed George Senior Applications Scientist, Environmental and Food Safety, Chromatography and Mass Spectrometry Thermo Fisher Scientific, Inc. Debadeep Bhattacharyya Senior Marketing Manager, Triple Quadrupole MS Thermo Fisher Scientific, Inc. Moderator: Laura Bush, Editorial Director, LCGC Sponsored by Presented by For questions, contact Kristen Moore at kmoore@advanstar.com

4 Figure 3: Schematic Mark-Houwink plots for three star polymers synthesized via core-first approach compared to a linear molecule. Arms are started from a multi-functional initiator core molar mass increase is a result of the addition of monomer units to the arms. The Mark-Houwink plots are parallel to one of the linear polymers but shifted to lower intrinsic viscosities. 3,0,5 Linear,0 log (η) 1,5 1,0 0,5 0,0 3 arm star 4 arm star 7 arm star -0,5-1,0 A very interesting observation is the sudden change of intrinsic viscosity occurring at approximately twice the molecular weight of the peak maximum. The drastic increase in viscosity is most probably a result of the formation of H-shaped molecules as coupling of two stars occurs via an arm log M In contrast to the application above, star polymers can also be synthesized using the core first approach, for example, using a multifunctional initiator. In this case the observations and results would be different. There would be no structural change because the molar mass increase of the star would result from the 6 A class of its own The Prominence series is perfect for LC, GPC and IC applications or as LC-MS front-end The Prominence LC-0A modular HPLC system combines optimum flexibility with increased productivity and performance. It provides the ultimate platform for user benefits in all application fields, such as pharmaceutical, (bio-)chemical, environmental and food analysis. Superior performance through powerful modules, merging productivity with best possible accuracy and reliability Customized hardware configuration provides solution-oriented systems for full flexibility, compatibility and expandability Flexible software control through Shimadzu LabSolutions LC/GC, LC-MS or DB/CS software, software packages from other vendors are also supported FDA 1 CFR Part 11 compliance for stand-alone workstation as well as network environments

5 Figure 4: Separation of a star branched polymer by interaction chromatography. The resolution of GPC/SEC for such samples is lower, because GPC/SEC separates based on size, which for this application increases only slightly for stars with more arms. Normalized concentration Arm/Precursor arms/dimer growth of the arms. For such star polymers the Mark Houwink plots are expected to be as shown in Figure 3. For each star polymer the Mark- Houwink plot will be shifted in parallel to lower viscosities at the same molar mass. Analysis would yield the same Mark-Houwink, α, but a reduced Mark-Houwink, K (intercept). The shift to lower intrinsic viscosities increases with increasing number of arms. Star with 3 Arms Elution volume (ml) Advanced Separation Techniques One limitation of GPC/SEC is that it separates only based on the size of the molecule in solution. This has the following consequences for branched samples: t$powfoujpobmdbmjcsbujpoxjuisfgfsfodf materials will underestimate the molar mass of the branched samples. A solution here is the use of molar mass sensitive detectors, such as on-line viscometers or light scattering detectors. PSS WinGPC scientific V & 10 The viscometer can then be used for structure analysis and for molar mass determination based on universal calibration. t*odbtftxifsfuifizespezobnjdwpmvnf increases only slightly with molar mass, such as arm-first star polymers, the resolution of size-based separations is limited. In this case interaction chromatography can be used as a complementary technique. Figure 4 shows the chromatogram of a gradient separation of an arm-first star polymer with a very high resolution even for stars with higher arm numbers. t*gtbnqmftfyijcjucspbenpmbsnbtt distributions besides structural heterogeneity (for example, branched and linear chains present) the risk of co-elution increases. In that case branched molecules of higher molar mass with the same hydrodynamic size as linear chains of lower molar mass elute at the same retention volume. Consequently, the GPC/SEC fractions eluting from the column cannot be regarded to be monodisperse any longer. Comprehensive characterization of branched polymers might be possible when D separations are applied. Two-dimensional combines two independent separation ULTRA-FAST GC Useful Tool or Just Another Gimmick? ON-DEMAND WEBCAST Aired May 31, 016 Register free at EVENT OVERVIEW The fundamental principle of ultra-fast gas chromatography is based on rapid temperature programming of the GC analytical column at rates generally between 60 and 00 C per minute. At these ramp rates, the dynamic temperature range of most columns is used up in less than minutes, and this is insufficient time to fully elute highboiling-point compounds, so the technique lends itself to short columns. Shorter columns have less resolving power, therefore it s important to know the techniques to optimize the parameters that give speed and resolution without sacrificing column capacity. Key Learning Objectives Where to use ultra-fast GC How to optimize parameters to get the best resolution without sacrificing column capacity Types of ultra-fast GC systems Who Should Attend GC Users with large numbers of samples looking to increase sample throughput GC Users with long analysis cycle times looking for faster cycle times GC Users looking to reduce energy usage and environmental impact of GC analysis Presenter Phillip James Managing Director Ellutia Ltd Sponsored by Moderator Meg L Heureux Managing Editor LCGC Presented by FREE executive summary for all webcast attendees. For questions, contact Kristen Moore at kmoore@advanstar.com

6 Figure 5: Separation of linear and comb shaped copolymers, which co-elute in GPC/ SEC alone. Gradient elution volume (ml) techniques to generate contour plots, which can also be used to quantify the different species. Figure 5 shows an example where linear and comb shaped molecules of different composition have been successfully separated. Summary t #SBODIJOHDBOPDDVSBTBTJEFSFBDUJPOJO polymerizations or can be introduced to tailor application properties. t 0OMJOFWJTDPNFUFSTQSPWJEFBDDFTTUPUIF SEC elution volume (ml) density of a molecule and can help to characterize branched molecules as they monitor structural changes with molar mass. t 1PMZFUIZMFOTFYIJCJUJOHMPOHDIBJOCSBODIFT can be analyzed by on-line viscometry, while information on short-chain branching can be gained by Fourier-transform infrared spectroscopy (FT-IR) detection. t $PFMVUJPOPGCSBODIFEBOEMJOFBS molecules can be a problem. In such cases advanced separation techniques or two-dimensional chromatography can be applied. References 1. D. Held, The Column 8(), 1 16 (01).. P. Montag, The Column 1, (008). 3. J. Gerber and W. Radke, Polymer 46, 4 (005) doi: /j. polymer Daniela Held studied polymer chemistry in Mainz, Germany. She currently works at the PSS software and instrument department and is responsible for education and customer training. Peter Montag studied chemistry at the University of Duesseldorf, Germany, achieving his PhD at the Max Planck Institute for Coal Research. He is the head of the PSS contract analysis department and responsible for hyphenated techniques. Wolfgang Radke studied polymer chemistry in Mainz, Germany, and Amherst, Massachusetts, USA. He is head of the PSS application development department and is also responsible for instrument evaluation and for customized training courses. Website: DHeld@pss-polymer.com